Alzheimer disease (AD) is a progressive neurodegenerative disorder that currently affects over 4.5 million Americans. By the middle of the century, the prevalence of AD in the USA is projected to almost quadruple. As current therapies do not abate the underlying disease process, it is very likely that AD will continue to be a clinical, social, and economic burden. Progress has been made in our understanding of AD pathogenesis by studying transgenic mouse models of the disease and by utilizing primary neuronal cell cultures derived from rodents. However, key proteins that are critical to the pathogenesis of this disease exhibit many species-specific differences at both a biophysical and functional level. Additional species differences in other as yet unidentified AD-related proteins are likely to also exist. Thus, there is an urgent need to develop novel models of AD that recapitulate the complex array of human proteins involved in this disease. Cell culture-based models that allow for rapid high-throughput screening and the identification of novel compounds and drug targets are also critically needed. To that end we propose to model both sporadic and familial forms of AD by generating two novel human embryonic stem cell lines (hES cells). Differentiation of these lines along a neuronal lineage will provide researchers with an easily accessible and reproducible neuronal cell culture model of AD. These cells will also allow high-throughput screening and experimentation in neuronal cells with a species-relevant complement of human proteins. In Aim 1 we will develop and characterize hES cell lines designed to model both sporadic and familial forms of AD. To model sporadic AD we will stably transfect HUES7 hES cells (developed by Douglas Melton) with lentiviral constructs coding for human wild type amyloid precursor protein (APP-695) under control of the human APP promoter. APP is well expressed within hES cells and upregulated upon neuronal differentiation. To model familial AD and generate cells that exhibit a more aggressive formation of oligomeric Aï¢ species we will also develop a second hES cell line stably transfected with human APP that includes the Arctic (E693G) mutation.In Aim 2 we will utilize our wild-type APP hES cells to perform a high-throughput siRNA screen. We will utilize AMAXA reverse-nucleofection in conjunction with a human druggable genome siRNA array (Dharmacon) that targets 7309 genes considered to be potential therapeutic targets. Following transfection conditioned media will be examined by a sensitive ELISA to identify novel targets that modulate Aï¢ levels. In addition a Thioflavin S assay will determine any effects on Aï¢ aggregation. Follow-up experiments will confirm promising candidates identified in the high-throughput screen. Taken together these studies aim to establish novel AD-specific hES cell lines and identify promising new therapeutic targets for this devastating disease.

Statement of Benefit to California:

Alzheimer disease (AD) is a progressive neurodegenerative disorder that currently affects over 500 thousand Californians. As the baby-boomer generation ages the prevalence of AD in California is projected to almost quadruple such that 1 in every 45 individuals will be afflicted. As current therapies do not abate the underlying disease process, it is very likely that AD will continue to be a major clinical, social, and economic burden. Some estimates have even suggested that AD alone may bankrupt the current Californian health care system. Progress has been made in our understanding of AD by studying rodent-based models of the disease. However, key proteins that are critical to the disease exhibit many species-specific differences at both a biophysical and functional level. Thus, there is an urgent need to develop novel models of AD that exhibit the complex array of human proteins involved in this disease. Cell culture-based models that also allow for rapid high-throughput screening and the identification of novel compounds and drug targets are also in critical need. The proposed studies aim to utilize human embryonic stem (hES) cells to establish a novel cell culture based model of Alzheimer’s disease. Once developed these cells will provide Californian researchers with a unique tool to investigate genes and proteins that influence the progress of AD. In this proposal we will also utilize these hES cells to perform a high-throughput screen of over 7300 genes to identify multiple novel drug targets that may critically regulate the development of this disease. Taken together these studies aim to establish novel AD-specific hES cell lines that can be utilized by multiple Californian researchers to identify promising new therapeutic targets for this devastating disease.

Review Summary:

SYNOPSIS: The PI’s overall aim is to develop a model of Alzheimer’s disease (AD) that recapitulates the complex array of human proteins involved in the disease to clarify the pathogenesis as well as provide a cell culture-based system for high-throughput screening for therapies. A rationale for the use of the hES cells is that the rodent Aβ is different biochemically and biophysically from human Aβ and does not form fibrils. In aim 1, the PI will stably transduce two hES cell lines with a lentiviral vector that contains human wild type or mutant APP ; these cells will then be differentiated towards a neuronal lineage and screened with an Aβ ELISA. In aim 2, the PI will utilize AMAXA nucleofection (or, alternatively, lentivrial shRNA delivery)of the hESCs to perform a high-throughput siRNA screen that targets 7,309 genes considered to be potential therapeutic targets, specifically attempting to identify targets that modify the generation or degradation of Aβ as determined by Aβ ELISA or affect Aβ aggregation as determined by thioflavin S fluorescence.

INNOVATION AND SIGNIFICANCE: This proposal is aimed at finding a cell culture model system for AD and to use this system for screening in order to identify new therapeutic targets in the disease. Considering the huge and growing problem of AD in the world, the proposal addresses a very significant world health problem. The need of having a reliable in vitro drug screening system (and an in vivo animal model) for screening for candidates drugs for AD is indisputable. At present, there are a number of cell culture models for AD, but they fall short for varied reasons, especially because they generally involve cell lines. The use of hESC-derived neuronal cells that express wild type or mutant APP will be of value since the cells will have a differentiated neuronal phenotype. In addition, the fact that these cells are human makes the cell culture model system a more relevant one than the use of cells from other species.

There were differing opinions on the innovativeness of the proposal. One reviewer found the idea of using an ES cell that expresses hAPP as a model system for AD and as a means of screening for therapeutic targets to be an original approach. In addition, the use of siRNAs to screen for the targets takes advantage of very contemporary cutting-edge technology. Another reviewer believed that expressing APP under its native promotor control does not equate to establishing a clone suitable for AD drug screening and found the innovative aspect of the proposed work is low.

STRENGTHS: The proposal investigates a significant health problem. The PI is a highly productive investigator who is highly recognized as a leader in the AD field. The collaboration of Dr. Loring, Director, Stem Cell Resource Co-Director, Human Embryonic Stem Cell Center, is a valuable one.

The proposal is well-written and well organized. The work contained in the proposal is ambitious but feasible. The PI addresses some potential problems that may arise and some alternative approaches. The lab is well qualified to analyze the amyloid precursor protein (APP) proteolytic products and has expertise in genetic modification of cells. It is likely that transfectants will be obtained. The use of the siRNA approach provides an unbiased screen for therapeutic targets. The work will draw upon knowledge gained from other model systems but will provide novel and useful information about the pathogenesis of AD.

WEAKNESSES: The proposal is a risky one; for example, the ELISA screening assay may not be sensitive enough to detect the small levels of Aβ in the siRNA screen described in specific aim 2. The PI notes a "personal communication" from Dr. Loring noting that APP is well expressed in hES cell lines and upregulated upon neuronal differentiation," but does not provide any supporting data.

The use of a differentiated hippocampal neuron or another neuronal cell type targeted in AD would be preferable to the more generic differentiated neuron - so that the results of this study may not be similar to those one would find in an AD transgenic mouse

One possible concern is that mutant APP will be toxic or have a specific phenotype preventing differentiation. In fact, some investigators have hypothesized that there is a failure in neurogenesis in Alzheimer's disease causing much of the symptomatology. However, a failure of the cells to differentiate may not be without interest.

Other concerns expressed included the lack of experience working with hESC and with high through-put screening especially involving siRNA transfection.

DISCUSSION: There was discussion on the cell target for siRNA transfection (hESC-derived neurons); on the sensitivity of the ELISA - think may run into trouble with heterogenity; and on the technical capability of the lab for this type of screening.

PROGRAMMATIC REVIEW: The Working Group voted to recommend this application for special consideration for funding should additional funds become available based on: 1) it was the only Alzheimer's disease-focused proposal; and 2)the applicant is a very accomplished researcher in this area.